1. Field of the Invention
The present invention relates to a new method of producing porcine Intrinsic Factor as well as to the protein produced using this method. In particular, the vector comprising the DNA sequence encoding the protein is introduced into a mammalian host cell, for example, a Chinese Hamster Ovary (CHO) cell, for a time and under conditions sufficient for optimal expression of the protein.
2. Background Information
Anemia is the major disorder related to low serum vitamin B12 levels. Megaloblastic anemia (MA), characterized by elevated mean corpuscular volume (MCV), has been found to be due to vitamin B12 deficiency. The relationship between vitamin B12 levels and MA is not always clear in that some patients with MA will have normal vitamin B12 levels; conversely, many individuals with vitamin B12 deficiency are not afflicted with MA. Despite these complications, however, in the presence of MA (e.g., elevated MCV) there is usually serum vitamin B12 deficiency. A major cause of vitamin B12 deficiency is pernicious anemia. This disease is characterized by poor vitamin B12 uptake, resulting in below normal serum vitamin B12.
There a number of conditions that manifest themselves as low serum vitamin B12 levels, including iron deficiency, normal near-term pregnancy, vegetarianism, partial gastrectomy/ileal damage, oral contraception, parasitic competition, pancreatic deficiency, treated epilepsy, and advancing age. Disorders associated with elevated serum vitamin B12 levels include renal failure, liver disease, and myeloproliferative diseases.
Intrinsic Factor binds vitamin B12. This characteristic enables the detection of and measurement of the quantity of vitamin B12 in biological samples. In conventional preparation of Intrinsic Factor, the Intrinsic Factor protein is isolated from porcine tissue by means of an expensive, tedious, and time-consuming process.
cDNA cloning using reverse transcriptase-polymerase chain reaction technique (RT-PCR) is well-known in the art. The designing of primers based on homology known for this particular protein in other species (such as human, mouse and rat) and selecting the appropriate PCR conditions to obtain cDNA require a significant amount of planning and expertise in the PCR-based cloning technique.
U.S. Pat. No. 3,591,678 discloses a process for purifying Intrinsic Factor by a batch chromatography process that utilizes an ion exchange resin. Impure Intrinsic Factor is dissolved in a buffer solution having relatively low pH and ionic strength, and the resultant solution is contacted with a cellulosic exchange resin. The resin is separated from the solution and the purified Intrinsic Factor is eluted therefrom with a buffer solution having a higher pH and ionic strength than the buffer solution in which the impure Intrinsic Factor was dissolved.
U.S. Pat. No. 4,447,528 discloses a radioassay procedure and reagent kit therefore for detecting auto-blocking antibody, such as auto blocking antibody which interferes with the complexation of Intrinsic Factor with vitamin B12. A receptor, i.e., Intrinsic Factor, is immobilized on a support and the amount of ligand, i.e., vitamin B12, capable of binding therewith in the presence of a biological fluid sample is determined.
U.S. Pat. Nos. 5,227,311 and 5,459,242 disclose a method for purifying an aqueous Intrinsic Factor solution which contains R-protein. The method involves adding to the Intrinsic Factor solution an amount of colloidal silica to disperse lipid emulsion, an amount of cobinamide sufficient to bind substantially all of the R-protein in the solution and an amount of an Intrinsic Factor affinity resin sufficient to bind the Intrinsic Factor in the solution, washing the bound cobinamide and the R-protein from the resin, eluting the Intrinsic Factor from the resin, and dialyzing the eluted Intrinsic Factor. Also disclosed is a kit for conducting an assay for cobalamins which includes a conjugate of microparticles and purified Intrinsic Factor.
U.S. Pat. No. 5,350,674 discloses a non-isotopic competitive assay for vitamin B12, utilizing Intrinsic Factor labeled with horseradish peroxidase, by coupling via heterobifunctional cross-linking agents. In addition, a method for stabilizing the resultant conjugates by pretreatment with N-ethylmaleimide is disclosed.
Prior investigators have disclosed the cDNA sequences encoding human Intrinsic Factor (Genbank Accession No. M63154), mouse Intrinsic Factor (Genbank Accession No. L24191) and rat Intrinsic Factor (Genbank Accession No. J03577). The cDNA sequence of the porcine Intrinsic Factor is disclosed in U.S. Published Patent Application No. 2006/0177872 of Abbott Laboratories. This application also discloses a method of producing porcine Intrinsic Factor by the use of E. coli cells.
Porcine Intrinsic Factor is typically isolated from the tissue of the duodenum of a hog (e.g., Sus scrofa). This isolation is a tedious, expensive, and time-consuming procedure, and the yields are low. The native Intrinsic Factor isolated by currently used procedures lacks consistency in its purity and in its resulting performance in an immunoassay. Therefore, it would be desirable to produce porcine Intrinsic Factor in large quantities and to isolate porcine Intrinsic Factor in a single-step affinity isolation process. Recombinant protein produced in this manner would have consistent performance in a diagnostic immunoassay.
All patents and publications referred to herein are hereby incorporated in their entirety by reference.